Nerve cell differentiation promoter

ABSTRACT

A nerve cell differentiation promoter which comprises a physiologically active substance NK175203 or a pharmacologically acceptable salt thereof as an active ingredient is provided. It is expected that the nerve cell differentiation promoter of the present invention is applicable to a medicine for dementia, a nerve cell protective medicine or a medicine for peripheral neuropathy caused by anticancer agents.

TECHNICAL FIELD

The present invention relates to a novel nerve cell differentiationpromoter which comprises a physiologically active substance NK175203 ora pharmacologically acceptable salt thereof as an active ingredient andwhich is expected to be usable as, for example, a medicine for dementia,a nerve cell protective medicine or a medicine for peripheralneuropathy.

BACKGROUND ART

The physiologically active substance NK175203 is a publicly knowncompound described in International Patent Publication No. W094/05679.

It is reported that this compound has an activity as a bone marrow cellproliferation promoter.

It has been proved in vitro that a nerve growth factor (hereinafterreferred to as NGF) elongates neurites, regulates the production of aneurotransmitter and exerts an effect on the regeneration of the nervecells of an aged animal Age, 8, 19 (1985)!. On the other hand, it isknown that when NGF is added to an established cell line PC12 which hasbeen cloned from a rat pheochromocytoma, the PC12 cell ceases theproliferation and differentiates into adrenergic neuron cells havingneurites. Because of these effects, NGF has recently attracted attentionas one of medicines for dementia. By using these cells, it has beenclarified that a fibroblast growth factor and interleukin 6 induce theelongation of neurites similarly to NGF. Recently, it has been reportedthat staurosporine, which is a low molecular weight substance derivedfrom a microorganism, also induces the elongation of neurites ShinkeiKagaku, 26, 200-220 (1987)!.

The staurosporine as described above is highly valuable in medicaltreatments, since it is a low molecular weight substance, different fromNGF. However, it is regrettably considered that there is room forfurther inventigation in the practical application of the same due toits high toxicity.

Under these circumstances, it is highly important in medical treatmentsto provide a low molecular weight substance which has a lower toxicityand exhibits the effect of elongating neurites at a low concentration.

DISCLOSURE OF INVENTION

As the results of extensive studies, the present inventors have foundout that a physiologically active substance NK175203 or apharmacologically acceptable salt thereof has an activity for promotingthe differentiation of nerve cells.

The present invention has thus been completed on the basis of thisfinding.

Accordingly, the present invention relates to a nerve celldifferentiation promoter which comprises the physiologically activesubstance NK175203 or a pharmacologically acceptable salt thereof as anactive ingredient together with pharmaceutically acceptable excipient(s)or carrier(s). The present invention further relates to a medicine fordementia, a nerve cell protective medicine and a medicine for peripheralneuropathy comprising the physiologically active substance NK175203 or apharmacologically acceptable salt thereof as an active ingredienttogether with pharmaceutically acceptable excipient(s) or carrier(s).

BRIEF DESCRIPTION OF DRAWING

FIG. 1 shows the effect of NK175203 on improving the caudal nervelatency increased by the administration of an anticancer agent.

FIG. 2 shows the effect of NK175203 on improving the sensory nervefunction (heat sensitivity) depressed by the administration ofAdriamycin (ADM). FIG. 2a shows the test result on the 72nd day from theinitiation of the experiment, while FIG. 2b shows the test result on the85th day from the initiation of the experiment.

BEST MODE FOR CARRYING OUT THE INVENTION

The physiologically active substance NK175203 to be used in the presentinvention can be produced and obtained in accordance with the methoddescribed in International Patent Publication No. W094/05679.

More specifically, it can be obtained by culturing a microorganismbelonging to a genus Streptomyces and producing the above-mentionedphysiologically active substance NK175203 (for example, FERM BP-4372) ina medium, accumulating the physiologically active substance NK175203 inthe culture and then collecting the same.

The physiologically active substance NK175203 which is the compound usedin the present invention may be in the form of a pharmacologicallyacceptable salt thereof. Examples of such a salt include salts withalkali metals such as sodium and potassium and salts with alkaline earthmetals such as calcium.

The nerve cell differentiation promoter of the present inventioncomprising the physiologically active substance NK175203 as an activeingredient promotes the differentiation of nerve cells, and is thereforeeffective for repairing and treating various damages to nerve cells.Thus the promoter is expected to be usable as, for example, a medicinefor dementia, a medicine for peripheral neuropathy caused by anticanceragents, diabetes, etc., or a nerve cell protective medicine.

When the compound of the present invention is to be used as a nerve celldifferentiation promoter, it is administered in the form of injections,oral preparations, suppositories, etc., either alone or as a mixturethereof with excipient(s) or carrier(s). As the excipients and carriers,selection is made of pharmaceutically acceptable ones, and the type andcomposition thereof are determined depending on the administration routeand administration manner. As a liquid carrier, for example, used may bewater, alcohols, animal and vegetable oils such as soybean oil, peanutoil, sesame oil and mineral oil, and synthetic oils. As a solid carrier,used may be, for example, sugars such as maltose and sucrose, aminoacids, cellulose derivatives such as hydroxypropylcellulose, and organicacid salts such as magnesium stearate.

In the case of an injection, it is generally desirable to usephysiological saline, various buffers, solutions of sugars such asglucose, inositol and mannitol or glycols such as ethylene glycol,propylene glycol and polyethylene glycol. It may be also possible thatthe compound of the present invention is dissolved, prior to theadministration, in an appropriate solvent for injection (for example, aliquid for intravenous administration such as sterilized water,physiological saline, a glucose solution, an electrolyte solution or anamino acid solution) together with an excipient such as a sugar, forexample, inositol, mannitol, glucose, mannose, maltose or sucrose, or anamino acid, for example, phenylalanine.

The content of this compound in the formulation is usually in the rangefrom 0.1 to 100% by weight, preferably from 1 to 98% by weight, thoughit widely varies from formulation to formulation. In the case of aninjection, for example, the content of the active ingredient is usuallyin the range from 0.1 to 30% by weight, preferably from 1 to 10% byweight. When orally administered, the compound is formulated togetherwith the above-mentioned solid or liquid carrier(s) into tablets,capsules, powders, granules, solutions, dry syrups, etc. The capsules,tablets, granules and powders generally contain from 5 to 100% byweight, preferably from 25 to 98% by weight, of the active ingredient.

The dose may be determined depending on the age, body weight andconditions of the patient, the purpose of the treatment, etc. Thetherapeutic dose is generally in the range of from 1 to 100 mg/kg·daywhen parenterally administered, and from 5 to 500 mg/kg·day when orallyadministered.

The compounds of the present invention are characterized by having a lowtoxicity and showing only a small accumulated toxicity when continuouslyadministered. When intraperitoneally administered at once to a mouse ina dose of 500 mg/kg, the compound of the present invention shows nosymptom of toxicity.

To further illustrate the present invention in greater detail, thefollowing Examples will be given. However, it is to be understood thatthe present invention is not restricted thereto so long as it does notdepart from the spirit and scope of the same.

EXAMPLE 1

Test of neurite elongation of PC12 cells by physiologically activesubstance NK175203

The results were judged on the basis of morphological changes inaccordance with the method of Green et al., Ann. Rev. Neurosi., 3, 353(1980).

More specifically, PC12 cells were inoculated into Dulbecco's modifiedEagle medium containing 10% of fetal bovine serum and 10% of horse serumin such a manner as to give a cell density of 1×10⁴ /ml and thenincubated overnight in a collagen-coated 96-well multiplate at 37° C.under 5% of CO₂. Next, a sample was added thereto and the morphologicalchanges were microscopically observed after one day.

As a result, the effective concentration of the physiologically activesubstance NK175203 on inducing the neurite elongation of the PC12 cellswidely varied from 20 to 2.0 μg/ml.

In the above method, the PC12 cells were exterminated after one day dueto the cytotoxicity of the physiologically active substance NK175203 ata concentration of 50 μg/ml. This concentration is 2.5 to 25 timeshigher than the effective concentration of the substance for promotingthe nerve cell differentiation, which means that the physiologicallyactive substance NK175203 has a relatively low toxicity.

EXAMPLE 2

Test of Physiologically active substance NK175203 on anticanceragent-induced peripheral neuropathy model

Examination was made on the effect of the physiologically activesubstance NK175203 on improving an anticancer agent-induced peripheralneuropathy model. Male CDF1 mice aged 5 weeks were employed as the testanimals. The neuropathy was induced by cisplatin (hereinafter referredto simply as CDDP) or Adriamycin (hereinafter referred to simply asADM). CDDP was intraperitoneally administered in a dose of 5 mg/kg onthe 0th, 7th, 14th, 21st, 28th and 32nd days from the initiation of theexperiment, while ADM was administered via the tail vein in a dose of 10mg/kg on the 0th, 4th and 14th days from the initiation of theexperiment. As a medicine for examining the improving effect, NK175203was used. NK175203 was administered thrice a week (Monday, Wednesday andFriday) for 6 weeks. The day of the initiation of the experiment (i.e.,the 0th day) was Monday. The drugs were each dissolved in physiologicalsaline before using. Table 1 summarizes the dose and administrationroute of each group. The caudal nerve latency was measured on the 0th to42nd days from the initiation of the experiment. A tail-flick test (aheat sensitivity examination) was carried out on the 72nd and 85th days.

                  TABLE 1                                                         ______________________________________                                        CDDP- or ADM-induced peripheral neuropathy model groups                             Neuropathy-                                                                             Medicine administered and                                                                        Number of                                  Group induction dose thereof       animals                                    ______________________________________                                        1     none      none               6                                          2     none      NK175203 40 mg/kg (intraperito-                                                                  5                                                          neally), thrice/week × 6 weeks                          3     CDDP      none               5                                          4     CDDP      NK175203 10 mg/kg (intraperito-                                                                  5                                                          neally), thrice/week × 6 weeks                          5     CDDP      NK175203 40 mg/kg (intraperito-                                                                  5                                                          neally), thrice/week × 6 weeks                          6     ADM       none               5                                          7     ADM       NK175203 10 mg/kg (intraperito-                                                                  5                                                          neally), thrice/week × 6 weeks                          8     ADM       NK175203 40 mg/kg (intraperito-                                                                  5                                                          neally), thrice/week × 6 weeks                          ______________________________________                                    

(i) Measurement of caudal nerve latency

The caudal nerve latency was measured in the following manner. A mousewas lightly etherized and fixed face upward onto a cork plate. As arecording electrode, a concentric electrode was penetrated into the tailon the peripheral side at a distance of 2 cm from the base. Further, twoneedle electrodes were inserted into a site 4 cm apart therefrom asstimulating electrodes. The stimulation was input from the stimulatingelectrode for a persistence time of 0.05 msec at 5 to 10 V via astimulation device (Model SEN-7203, mfd. by Nippon Kohden Corporation)and an isolator (Model SS-320J, mfd. by Nippon Kohden Corporation). Theaction potentials, which were obtained via the recording electrodes andan amplifier (Model EI-601G, mfd. by Nippon Kohden Corporation), of 20to 50 stimulations were added up. Then the average was calculated andrecorded with an X-Y recorder (Model WX-2400, mfd. by Graphtec Corp.).The latent time of the action potential thus obtained was divided by thedistance between the stimulated site and the recording site to therebygive the conduction velocity.

On the 42nd day from the initiation of the experiment, the caudal nervelatency was measured. FIG. 1 shows the results. Compared with the intactgroup, the group of the administration of CDDP alone showed asignificantly reduced conduction velocity, which indicated thatneuropathy had been induced. On the other hand, the groups of thecombined use of CDDP with NK175203 showed each a significantly increasedconduction velocity compared with the group of the administration ofCDDP alone. When the decrease of the velocity due to the administrationof CDDP is taken as 100%, the groups of the combined use of CDDP with 10and 40 mg/kg of NK175203 showed the improving effects of 45 and 60%respectively in a dose dependent manner.

(ii) Tail-flick test (heat sensitivity examination)

The tail-flick test was carried out in the following manner. A thermallamp of a Natsume Thermal Counter (mfd. by Natsume) irradiated at 6 V.After preliminarily heating the measuring board by irradiating with thelamp for 30 seconds, the lamp was switched off. Then a mouse set in acircular cylinder for fixation was quickly placed under the lamp. Thenthe tail of the mouse was irradiated with the thermal lamp about 10 cmabove and the time required until the mouse sensed heat and flicked itstail was measured.

On the 72nd day from the initiation of the experiment, the groups ofADM-induction models were subjected to the first tail-flick test. Thesite 4 cm apart from the tail base was irradiated with the thermal lamp.FIG. 2a shows the results. Compared with the group of the administrationof ADM alone, the group with the combined use of ADM with 40 mg/kg ofNK175203 showed a significantly shortened flick time, thus indicating animprovement in the response to the heat stimulation.

Next, the second test was conducted on the 85th day. The site 3 cm apartfrom the tail base was irradiated with the thermal lamp. FIG. 2b showsthe results. Compared with the intact group, the group with theadministration of ADM showed a significantly delayed flick time, thusindicating that the response to the heat stimulation had been depressedby the administration of ADM. Further, the groups with the combined useof ADM with NK175203 showed flick times similar to that of the intactgroup, thus indicating that the sensory nerve function depressed by theadministration of ADM had been clearly ameliorated.

Based on these results, it has been confirmed that NK175203 exerts animproving effect on the mouse anticancer agent-induced peripheralneuropathy models.

EXAMPLE 3 (Formulation Example 1)

Production of granules:

Fifty parts by weight of sodium salt of the physiologically activesubstance NK175203, 600 parts by weight of lactose, 330 parts by weightof crystalline cellulose and 20 parts by weight ofhydroxypropyl-cellulose were well mixed together. Then the obtainedmixture was compressed by using a roll compressor (Roller Compactor™),ground and dressed through 16-mesh and 19-mesh sieves to thereby givegranules.

EXAMPLE 4 (Formulation Example 2)

Production of tablets:

One hundred parts by weight of sodium salt of the physiologically activesubstance NK175203, 90 parts by weight of crystalline lactose, 107 partsby weight of crystalline cellulose and 3 parts by weight of magnesiumstearate were mixed in a V-type mixer and then the obtained mixture wasshaped into tablets each weighing 300 mg.

EXAMPLE 5 (Formulation Example 3)

Production of injection:

To 50 parts by weight of sodium salt of the physiologically activesubstance NK175203 and 120 parts by weight of mannitol was addeddistilled water so as to give a total volume of 2,000 parts. Afterdissolution, the solution was sterilely filtered through a milliporefilter of GS type. The filtrate was introduced in 2-g portions into 10ml vials and freeze-dried to thereby give a freeze-dried powder forinjection containing 50 mg/vial of the sodium salt of the compound ofthe present invention.

INDUSTRIAL APPLICABILITY

As described above, the physiologically active substance NK175203 or apharmacologically acceptable salt thereof has an activity foraccelerating the elongation of neurites of PC12 cells and a relativelylow toxicity. Thus it is useful as a nerve cell differentiation promoterand expected to be applicable to, for example, a medicine for dementia,a medicine for peripheral neuropathy caused by anticancer agents,diabetes, etc., or a nerve cell protective medicine.

We claim:
 1. A method for promoting nerve cell differentiationcomprising administering an effective amount of the substance NK175203or a pharmacologically acceptable salt thereof together with apharmaceutically acceptable excipient or carrier to an individual inneed thereof.
 2. A method for treating peripheral neuropathy comprisingadministering an effective amount of the substance NK175203 or apharmacologically acceptable salt thereof together with apharmaceutically acceptable excipient or carrier to an individual inneed thereof.